Mitochondrial damage, oxidative stress, and accumulation of Lewy bodies (LB) - are cardinal features of dopaminergic neuron injury in Parkinson's disease (PD). Neither mechanisms realizing free radicals into release of cell death factors, nor specific pathways leading to LB production are well understood. Damage of mitochondria by oxygen radicals can release cyt c thus threatening the integrity of the entire neuron. How neurons protect themselves against inadvertent release of death-signals from damaged mitochondria is not known. Our project will identify specific biochemical mechanisms and causative relationships between these essential PD-relevant features and their role in cell damage and death. Recently, we discovered that, during programmed cell death in non-neuronal cells, interaction of a mitochondria-specific phospholipid, cardiolipin (CL), with cyt c converts it into a CL-specific peroxidase essential for release of cell death factors. Our Preliminary results show that alpha-synuclein (Syn), binds anionic lipids and cyt c into a triple PEROXIDASE complex, that cross-links its components into LB-like hetero-oligomers. Covalent conjugation of Syn with cyt c prevents death signaling effects of cyt c in the cytosol but contributes to the formation of LB and Lewy neurites (LN). This protection from acute neuronal cell death does not come wdthout a price. Because the peroxidase activity is retained in aggregates of cyt c vth Syn/lipids, they could act as a source of oxidative stress, contributing to chronic neurodegeneration. This explains paradoxical data implicating Syn and LB in both protective and damaging roles in PD. Our three Specific Aims will: i) determine mechanisms of cyt c interactions with CL resulting in CL peroxidation in mitochondria of SH-SY5Y and MN9D cells treated with DA or rotenone and investigate the role of CL peroxidation products in release of cyt c from mitochondria into the cytosol. Novel mitochondria-targeted regulators of CL/cyt c peroxidase activity will be tested as inhibitors of CL peroxidation and neuroprotectors. 2) investigate peroxidase mechanisms of cross-linking of cyt c, Syn, and anionic phospholipids in Syn aggregation and preventing cell death. 3) utilize a rotenone-infusion rat model of PD to determine the extent to which interactions of peroxidase within a triple complex Syn/cyt c/anionic lipids are realized in vivo and extend these findings to analysis of Syn/cyt c interactions in dopaminergic neurons in the brain samples of patients with PD/ dementia with Lewy Bodies (DLB).